The roles of protein phosphatases in neuronal function and dysfunction are poorly understood. Protein phosphatase 2A (PP2A) is a ubiquitous and abundant enzyme with roles in most cellular processes that involve reversible Ser/'rhr phosphorylation. In neurons, PP2A dephosphorylates a staggering array of substrates include cytoskeletal proteins, receptors and ion channels, neurotransmitter-synthesizing enzymes, kinases, and transcription factors. PP2A is a trimeric holoenzyme of two core subunits (a catalytic and a scaffold subunit) complexed to a variable, regulatory subunit. The identity of the regulatory subunit determines where the PP2A holoenzyme is localized, which substrates it dephosphorylates, and how its activity is modulated by intracellular signaling. None of this has been investigated in any detail. Some variable, regulatory subunits are only expressed in brain, suggesting that brain-specific PP2A holoenzymes have unique roles in neuronal physiology and development. A trinucleotide repeat expansion in the promoter region of one of these neuron-specific subunits, B[beta], has recently been linked to spinocerebellar ataxia type 12 (SCA12). Thus, aberrant PP2A subunit expression may be a hallmark of certain human neurodegenerative disorders. In this proposal, we explore the roles of the brain regulatory subunit B[gamma] in subcellular localization of PP2A holoenzymes and developmental signal transduction cascades. Aim 1 investigates the sequence determinants for differential subcellular localization of B subunits, and for association with the two core PP2A subunits. Proteins that interact with the B[gamma] subunit are identified in Aim 2 using yeast two-hybrid and protein purification techniques. Aim 3 investigates the function of neuronal PP2A regulatory subunits in Ras-MAP kinase signaling and neurite outgrowth of PC12 cells and primary neurons. These studies will significantly advance our understanding of how the ubiquitous PP2A enzyme regulates specifically neuronal functions, and may lead to the development of drugs that target neuronal PP2A isoforms.